1. Field of the Invention
The invention relates generally to the field of diagnostics and detection. More particularly, the invention relates to low resource processors for assessing molecular interactions. Specifically, the invention relates to the use of devices containing multiple chambers separated by surface tension valves for the processing of microbeads having screening reagents attached thereto. The device permits assaying for the content of a wide variety of environmental and biological samples. In addition, specific interactions such as DNA-DNA interactions, DNA-protein interactions, or protein-protein interactions, ion-protein interactions, enzyme-substrate interactions can be assessed.
2. Discussion of the Related Art
Recent research has focused on the development of nucleic acid-based detection for low resource settings (Niemz et al., 2011). Nucleic acid-based detection systems, such as quantitative PCR (qPCR), are particularly attractive technologies for detection of pathogens because of their sensitivity, specificity and relatively rapid time-to-answer. The effectiveness of PCR is dependent on both the quality and quantity of nucleic acid template (Beuselinck et al., 2005) and the absence of interferents (Radstrom et al., 2004). For example, carbohydrates, proteins, lipids or other unidentified interferents present in clinical samples have all been shown to inhibit PCR and produce false negatives (Monteiro et al., 1997; Wilson, 1997; Coiras et al., 2003). In addition to various interferents, patient samples also contain nucleases, which directly reduce the number of nucleic acid targets present (Wilson, 1997).
To minimize false negatives and maximize the efficiency of nucleic acid-based diagnostics, nucleic acids are extracted and concentrated into an interferent-free buffer prior to testing. One classic laboratory method uses a phenol-chloroform cocktail (Chomczynski and Sacchi, 1987). This method is highly effective, but is not as commonly utilized today because it is time consuming and requires the use of toxic organic chemicals. Several solid phase extraction kits are commercially available to purify DNA or RNA from patient samples. Many of these kits rely on selective nucleic acid binding to silica-coated surfaces in the presence of ethanol and a chaotropic agent such as guanidinium thiocyanate (GuSCN) (Avison, 2007; Yamada et al., 1990). GuSCN also denatures protein contaminants including nucleases that may be present in the sample (Chirgwin et al., 1979; MacDonald et al., 1987). These kits are not cost effective for low resource use and often require the use of specialized laboratory equipment, such as a robot or centrifuge, and trained technicians that are unavailable in a low resource setting. Additionally, many involve multiple steps that increase the chance of contamination of both the sample and operator.
Microfluidics is one promising format for low resource nucleic acid-based diagnostics. Recently, there has been a growing interest in expanding microfluidic technologies for sample preparation (Niemz et al., 2011; Price et al., 2009). Many of these devices are suitable for integrating with downstream nucleic acid amplification and detection technologies (Chen et al., 2010; Hagan et al., 2011). However, the small surface area of solid phase available for nucleic acid binding and the limited sample volume that can be flowed through the channels limit the total mass of nucleic acid recovered (Niemz et al., 2011), and therefore negatively impact the limit of detection.
For example, the analysis of transrenal DNA for diagnosis of TB is an attractive alternative to traditional diagnostic methods such as sputum microscopy, especially as nucleic acid-based technologies become increasingly more relevant in low resource settings (Huggett et al., 2009). PCR analysis of patient urine samples is ineffective for some of the same reasons noted above. It is thought that one of the principle reasons is the high variation in salts contained in urine samples. However, the collection of urine is thoroughly noninvasive. This is particularly advantageous in high disease burden settings where limited resources and lack of skilled technicians may make sample collection difficult (Umansky and Tomei, 2006; Green et al., 2009). Additionally, the collection of urine provides a much larger sample volume than other more invasive methods such as sputum collection. While transrenal DNA is present in relatively low concentrations (˜6 to 50 ng/mL in healthy subjects (Bryzgunova et al., 2006)), the ability to collect and test a large volume ensures that there is sufficient material available to make an accurate diagnosis. Finally, the analysis of purified transrenal DNA can be multiplexed to detect the presence multiple pathogens simultaneously. For example, coinfection of TB with HIV is a common problem in Sub-Saharan Africa (Green et al., 2009). A multiplex detection assay designed to diagnose both TB and HIV would be ideal this setting.
The concept of transrenal DNA analysis for the diagnosis of tuberculosis is emerging as an attractive alternative to traditional diagnostic methods, particularly as nucleic acid-based technologies become increasingly more relevant in low resource settings. As dying human cells and microorganisms are broken down, small nucleic acid fragments can end up in the blood stream, and subsequently pass through the kidneys and into the urine (Botezatu et al., 2000). The analysis of transrenal DNA fragments for mycobacterial DNA has been demonstrated to be a promising technique for the diagnosis of tuberculosis and offers several advantages over traditional sputum microscopy (Aceti et al., 1999; Cannas et al., 2008; Gopinath and Singh, 2009). The collection of urine is thoroughly noninvasive. This is particularly advantageous in high disease burden settings where limited resources and lack of skilled technicians may make sample collection difficult (Umansky and Tomei, 2006; Green et al., 2009). Additionally, the collection of urine provides a much larger sample volume than sputum collection. While transrenal DNA is present in relatively low concentrations (˜6 to 50 ng/mL in healthy subjects (Bryzgunova et al., 2006)), the ability to collect and test a large volume ensures that there is sufficient material available to make an accurate diagnosis. Finally, the analysis of mycobacterium-specific DNA sequences is inherently more sensitive than microscopic diagnosis (Huggett et al., 2009).
In the absence of appropriate methodologies, sputum microscopy remains the current worldwide standard for the diagnosis of tuberculosis. Unfortunately, the collection of sputum samples can potentially produce infectious aerosols, which is particularly problematic in low resource settings where trained personnel are frequently unavailable (Cannas et al., 2008). Microscopic diagnosis is time consuming and suffers from limited sensitivity (Green et al., 2009). However, because methods for purifying DNA from urine in low resource environments prior to any nucleic acid-based diagnostic test are not available, this remains the preferred approach.
Similar issues relate to the testing for other molecular species including proteins, lipids and carbodydrates. In general, detection of any molecular species of interest is made much more difficult by the presence of interferents contained in a “real” sample. Therefore, a rapid, noninvasive diagnostic technology is desirable, especially in low resource environments.